Modulation of zinc levels to improve tissue properties

ABSTRACT

Methods of altering properties of tissues, by providing an effective modifying amount of zinc, which may be in the form of zinc ions. This is accomplished through the use of topical compositions containing one or more zinc-containing components such as salts and/or other zinc compounds or complexes, particularly zinc acetate. Altering properties of tissues includes increasing or decreasing fatty tissue, increasing or decreasing epidermal thickness, increasing elastin content, and preventing or treating gum regression or atrophy. Also disclosed is the provision of a contact lens coated with a zinc-containing material, to improve vision by increasing elastin content of the lens of the eye through release of zinc ions.

CROSS-REFERENCES TO RELATED APPLICATIONS

This Application claims priority from U.S. provisional patentapplication 60/421,278 filed Oct. 25, 2002, entitled “ImplantableMedical Devices Using Zinc” and U.S. provisional patent application60/421,336 filed Oct. 25, 2002, entitled “Modulation of Zinc Levels toImprove Tissue Properties”. This application is also related to U.S.non-provisional application entitled “Implantable Drug Devices UsingZinc”, of the same inventors filed on the same day herewith (attorneydocket 020154-001210US). The entire contents of all said applicationsare hereby incorporated herein.

BACKGROUND OF THE INVENTION

This invention relates to the use of zinc-containing compositions forpharmaceutical and cosmeceutical purposes.

Zinc is one of the most important trace elements in human health andnutrition and plays a significant role in the function of manyintracellular proteins. Zinc is crucial for gene expression and nucleicacid metabolism, which accounts in part for its importance in cellulargrowth and differentiation. Recent investigations indicate that zinc mayactually have a regulatory role. Zinc possesses ligand-bindingproperties that are utilized effectively at the catalytic site of abroad range of enzymes. In addition, it has many structural roles inbiological membranes [Tang et al., (2001) J Nutr 131: 1414-14200], cellreceptors, and proteins (i.e. transcription factors and proteinsinvolved in DNA replication). Zinc has been shown to have an effect onepidermal growth factor (EGF)-stimulated intracellular signaling [Wu etal., (1999) Am J Physiol 277: L924-L931] and numerous studies alsoindicate that zinc possesses insulin-like effects [Tang et al., supra].Zinc has also been shown to cause an increase in IGF-I and TGF-beta 1 infemoral-diaphyseal and metaphyseal tissue cultures [Ma et al. (2001) IntJ Mol Med 8(6): 623-8].

Studies have shown that zinc has an effect on epidermal growth factor(EGF) stimulated signaling. Addition of 0.3 mM of zinc or epidermalgrowth factor resulted in a marked increase in tyrosine phosphorylationof proteins in whole cell extracts [Hansson et al. (1996) Arch Biochemand Biophys 328 (2): 233-238]. Zinc has been found to be a potentinhibitor of protein tyrosine phosphatase (PTPase) [Wang et al. (1992) JBiol Chem 267(23): 16696-16702], which may induce increased proteintyrosine phosphorylation and generate activation of a host ofintracellular signaling that includes MAP kinase activity [Hansson etal., supra]. A wide range of integrated biological responses has beenassociated with EGFR signaling. These biological responses includemitogenesis, apoptosis, enhanced cell motility, protein secretion, anddifferentiation or dedifferentiation even on the same cell, depending onthe phenotype [Wells (1999) EGF receptor. Int J Biochem Cell Biol 31(6):637-643]. EGFR signaling in adult animals has been postulated to play arole in organ repair and experimental results indicate that EGFRinhibition affects epithelial cell proliferation and stratification.Furthermore, EGFR may also affect wound healing and play a role inmaintaining normal epithelial thickness [Nakamura et al. (2001) Exp EyeRes 72(5): 511-517]. In a number of patents and publications, zinc hasbeen implicated to play a role in increasing wound healing, and the EGFRsignaling pathway may be a key to its ability to help repair wounds.

Zinc has the potential to exert insulin-like effects with respect tolipogenesis [Coulston et al. (1980) Diabetes 29(8): 665-667], glucosetransport and glucose oxidation in rat epididymal adipocytes [Shishevaet al. (1992) Diabetes 41(8): 982-988; May et al. (1982) J Biol Chem257(8): 4362-4368]. Moreover, zinc also potentiates the mitogenicsignaling of insulin [Kiss et al. (1997) FEBS Lett 415(1): 71-74].Evidence points out that zinc may actually be involved in several stepsof the insulin-signaling pathway. Zinc has been reported to exertpositive effects on insulin synthesis and secretion and also is requiredfor structural conformation of insulin [Chausmer et al. J Am Coll Nutr17: 109-115]. In adipocytes, zinc has been shown to stimulateinsulin-specific binding through an unknown mechanism [Gomot et al.(1992) Biol Trace Elem Res 32: 331-335; Herington (1985) Horm Metab Res17: 328-332]. As mentioned above, zinc also possesses the ability toinhibit PTPase activity. PTPase is an early and critical juncture ininsulin signaling. Membrane-associated PTPase activity antagonizes theeffects of the insulin receptor and other growth factor-associatedtyrosine kinases [Kremerskothen et al. (1993) Mol Cell Biochem 125: 1-9;Li et al. (1997) Endocrinology 138: 2274-2279; Samet et al. (1999) Am JRespir Cell Mol Biol 21: 357-364]. There are several branch points inthe insulin-signaling pathway. One of these branch points involves theactivation of phosphatidylinositol 3-kinase (PI 3-kinase). PI 3-K iswell known to be necessary for the recruitment of GLUT4 to the cellsurface. Specific isoforms of protein kinase C (PKC) appear to benecessary for the redistribution of GLUT 4 from intracellular storagesites to the plasma membrane [Braiman et al. (1999) Mol Endocrinol 13:2002-2012; Kanoh et al. (2000) J Biol Chem 275: 16690-16696; Standaertet al. (1999) J Biol Chem 274: 25308-25316.]. PKC membrane localizationand activity can be stimulated by zinc [Csermely et al. (1988) J BiolChem 263: 6487-6490; Forbes et al. (1990) Biochem Int 22: 741-748;Forbes et al. (1990) Biochim Biophys Acta 1053: 113-117; Quest et al.(1992) J Biol Chem 267: 10193-10197]. Recent evidence has shown thatzinc may regulate the Ser/Thr protein kinase termed mammalian target ofrapamycin or mTOR (also known as FRAP and RAFT) [Lynch et al. (2001) AmJ Physiol Endocrinol Metab 281(1): E25-E34]. The mTOR signaling pathwaybegins at the PI 3-kinase activation site. A downstream target of themTOR pathway is the 40 S ribosomal protein S6, which is a substrate ofp70^(S6k) [Lynch, supra]. Amino acids increase mRNA translation(independently of merely serving as substrates for synthesis) throughribosomal protein S6 kinase [Patti et al. (1998) J Clin Invest 101(7):1519-1529]. The 70 kDa ribosomal S6 kinase (p70^(S6K)) is an importantregulator of cellular translational capacity due to its ability tophosphorylate the 40 S ribosomal protein S6 and regulate 5′-terminaloligopyrimidine tract mRNAs [Martin et al. (2001) J Biol Chem 276(11):7884-7791]. The activation of ribosomal protein S6 thereforeup-regulates ribosome biosynthesis and enhances the translationalcapacity of the cell. Additionally, ribosomal protein S6 has beenimplicated in the regulation of cell size [Martin et al., supra]. 1061Insulin-like growth factor-I (IGF-I) and transforming growth factorbeta-1 (TGF-β1) play important roles in the biological system. Theeffect of zinc on IGF-I and TGF-β production was investigated todetermine the role of this metal on growth of growth in newborn rats[Ma, supra]. The results of the experiments showed that the presence ofzinc caused a significant increase in protein, IGF-I and TGFβ1concentrations in medium cultured with diaphyseal or metaphysealtissues. In addition, expression levels of IGF-I and TGF-β1 were alsosignificantly increased in the diaphyseal and metaphyseal tissuescultured with zinc. Transforming growth factor betas are multifunctionalpolypeptide growth factors that are involved in proliferation anddifferentiation of cells, embryonic development, wound healing andangiogenesis [Blobe et al. (2000) N Engl J Med 342(18): 1350-1358].Usually, TGF-beta1 is bound to the extracellular matrix, and can bereleased by proteases [Taipale et al. (1992) J Biol Chem 267:25378-25384]. The presence of extracellular matrix has been found todown regulate the expression of the TGF-beta1 gene [Streuli et al.(1993) J Cell Biol 120: 253-260]. Therefore, TGF-beta may act as afeedback regulator of extracellular matrix formation. TGF-β regulatescellular processes through binding to high-affinity membrane receptors,which causes the assembly of a receptor complex that phosphorylates theproteins of the SMAD family [Blobe et al., supra]. SMADs act as signaltransducers of TGF-β family members. After phosphorylation, SMADs form acomplex and move into the nucleus and assemble complexes that directlycontrol gene expression through DNA binding and recruitment oftranscriptional co-activators or co-repressors [Massague J. (2000) NatRev Mol Cell Biol 1(3): 169-178]. SMADs help to regulate a number ofgenes including those for collagen [Zhang et al. (2000) J Biol Chem275(50): 39237-39245] and regulation of SMADs is achieved in severaldifferent ways. Once in the nucleus, the activated SMAD complex mayactivate or repress gene expression. SMADs may bind to p300(co-activator) or TG3-interacting factor (TGIF) (co-repressor) dependingon their relative levels in a cell [Massague, supra]. Evidence suggeststhat TGIF may set the maximal level to which TGF-β signaling canactivate transcription [Wotton et al. (1999) Cell 97(1): 29-39].Signaling through the extracellular-signal-regulated kinase (ERK)increases TGIF levels [Lo et al. (2001) EMBO J 20(1-2): 128-136.]. ERKis a member of the mitogen-activate protein kinase (MAPK) pathway, whichmay be activated through activation of the EGF receptor pathway.

Zinc also has been shown to inhibit aggregation of platelets,particularly in a combined effect with plasma, and specifically withfibrinogen [Chvapil et al. (1975) Life Sciences (16): 561-572; Sauvageet al., U.S. Pat. No. 5,401,730].

Elastin is a resilient connective tissue protein present in theextracellular matrix and is especially abundant in tissues that undergorepeated physical deformations, i.e. lungs, blood vessels and skin[Parks (1997)]. Posttranscriptional regulation of lung elastinproduction. Am J Respir Cell Mol Biol 17: 1-2]. Elastin is a polymercomposed of enzymatically cross-linked tropoelastin, which is thesecreted soluble precursor protein [Zhang et al. (1999) Mol Cell Biol 9(11): 7314-7326]. Similar to other structural extracellular matrixproteins, the majority of elastin production is restricted to a narrowwindow of development. In the majority of tissues, elastogenesisincreases dramatically during late fetal life, peaks near birth andearly neonatal life, decreases significantly soon after and is nearlyrepressed by maturity. Previous investigations have shown thatinsulin-like growth factor-I (IGF-I) increased elastin genetranscription through displacement of protein binding to the proximalpromoter. Sp 1 and Sp3 have been identified as factors whose binding isabrogated by IGF-I [Conn et al. (1996) J Biol Chem 271(46):28853-28860]. At the post-transcriptional level, TGF-β increases elastingene expression through increasing the stability of tropoelastin mRNA[Kahari et al. (1992) Lab Invest 66(5): 580-588]. Zinc affects IGF-I andTGF-β expression, which suggests that zinc may increase elastinproduction through these two proteins. After tropoelastin synthesis, a67 kDa elastin binding protein binds it and acts as an effectivechaperone, preventing its premature intracellular aggregation [Hinek etal. (1994) J Cell Biol 126(2): 563-574]. Tropoelastin and the elastinbinding protein remain bound until the complex is excreted into theextracellular space where the chaperone interacts with galactosugars ofthe microfibrils, decreasing its affinity for the tropoelastin molecule.Microfibrillar components act as scaffolds for the deposition ofelastin. Once the tropoelastin molecules are properly aligned, they arecross-linked by lysyl oxidase [Robert (1999) Connect Tissue Res 40 (2):75-82]. A combination of ascorbic acid, tyrosine, and zinc sulfateapplied to the skin has been shown to produce a readily observablediminution of the fine wrinkle structure [Schinitsky et al., U.S. Pat.No. 4,938,969]. The mechanism was not clearly understood, but the patentstates that the three ingredients were believed to function incooperation to stimulate fibroblast proliferation and to promote theirproduction of collagen and elastin, thereby promoting the supportingrole of the associated dermal tissue (col. 2 lines 12-16).

Other patents disclose beneficial effects of certain zinc compounds whencombined with other active agents on skin. Thornfeldt, U.S. Pat. No.6,071,543 discloses combinations of salts of pyridinethiol oxides andcombinations of such salts with metal oxides and thiols, to treat orprevent signs of aging in skin or mucous membranes. Specificcombinations mentioned include zinc pyrithione with selenium pyrithioneand zinc pyrithione with selenium sulfide. Perricone, U.S. Pat. No.5,554,647 includes a discussion of the use of zinc (for example, in theform of zinc sulfate) as a secondary ingredient in compositions fortreatment of aging skin, where the primary active ingredient in thecompositions is an acetylcholine precursor. The zinc is said to beeffective for enhancement of neurotransmitter synthesis. Murad, U.S.Pat. No. 5,972,999, discloses compositions for skin treatment whoseprimary active ingredient is one or more sugar compounds that areconverted into glycosaminoglycans in the bloodstream. These compositionsmay also include a zinc component, preferably zinc complexed with anamino acid such as methionine Such zinc compounds are said to assist insome way in binding collagen and elastic tissue in order to rebuilddamaged or aged skin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a)-1(d) depict photomicrographs of murine skin sectioned andstained, treated with varying concentrations of zinc ion to increaseelastin content.

FIG. 2 depicts photomicrographs of murine skin sectioned and stained,treated with a composition containing zinc, and a control, demonstratingtropoelastin content.

FIG. 3 is a graphical depiction of increase in tropoelastin contentmurine skin using a composition of the invention.

FIGS. 4-6 are graphical depictions of comparative scores for elastincontent, epidermal thickness and hypodermal fat, for a composition ofthe invention as compared to a control.

FIGS. 7-11 are depictions of test results on increase in skin elasticityby surface application of compositions of the invention, using graphsand photographs.

FIG. 12 is a graphical depiction of cross sectional elastin contentafter a 21-day topical application of zinc or control.

FIG. 13 contains graphical depictions of information from experimentsconducted to investigate the effects of concentration on elastinproduction.

SUMMARY OF THE INVENTION

The present invention relates to zinc-containing compositions andmethods, particularly topical methods, which are employed to achieve oneor more of several effects, including increasing or decreasing epidermalthickness, increasing elasticity or elastin levels, and increasing ordecreasing fat in or near the skin. While we do not wish to be boundthereby, it is believed that these formulations and methods provide suchresults by supplying increased local ionic zinc concentrations to therelevant tissues.

In general, the invention comprises a method for altering the propertiesof tissue of a subject, said altering being selected from the groupconsisting of increasing fat content in said tissue, decreasing fatcontent in said tissue, increasing epidermal thickness, decreasingepidermal thickness, and increasing elastin content in said tissue, saidmethod comprising applying a dermatologically or pharmaceuticallyacceptable composition consisting essentially of one or morezinc-containing components (e.g. zinc compounds, complexes and/orchelates) in admixture with a dermatologically or pharmaceuticallyacceptable carrier, in an effective tissue-modifying amount to one ormore sites on said tissue in need of said modifying.

More specifically, in one aspect the invention comprises a method forincreasing fat production in the skin (i.e., “superficial fat”) orbeneath the skin (i.e., “deep fat”) of a subject, which comprisestopically applying a dermatologically or pharmaceutically acceptablecomposition consisting essentially of one or more zinc-containingcomponents in admixture with a dermatologically or pharmaceuticallyacceptable carrier, in an amount effective to increase fatty tissue, toa site on the subject in need of increased fat. In another aspect theinvention comprises a method for decreasing fat production in or beneaththe skin, which comprises topically applying a dermatologically orpharmaceutically acceptable composition consisting essentially of one ormore zinc-containing components in an amount effective to decrease fattytissue to a site on the subject in need of decreased fat. By increasingor decreasing fatty tissue is meant to increase or decrease the mass offatty tissue, which may be done by affecting either the number ofadipocytes or the size of individual existing adipocytes. In a thirdaspect the invention comprises a method for increasing elastin contentin a tissue of a subject (including but not limited to the skin), whichcomprises topically applying to said tissue a dermatologically orpharmaceutically acceptable composition consisting essentially of one ormore zinc-containing components in admixture with a dermatologically orpharmaceutically acceptable carrier in an effective elastin-increasingamount. A fourth aspect of the invention comprises a method forincreasing epidermal thickness in a subject, the method comprisingtopically applying a dermatologically or pharmaceutically acceptablecomposition consisting essentially of one or more zinc-containingcomponents in admixture with a dermatologically or pharmaceuticallyacceptable carrier in an effective epidermal thickness-increasingamount, to an area of the skin of the subject in need of increasedepidermal thickness. In another aspect, the invention comprises a methodfor decreasing epidermal thickness in a subject, which comprisestopically applying a dermatologically or pharmaceutically acceptablecomposition consisting essentially of one or more zinc-containingcomponents in admixture with a dermatologically or pharmaceuticallyacceptable carrier to an area of the skin of the subject in need ofdecreased epidermal thickness.

Yet another aspect of the invention comprises a method for increasingelastin content of the lens of an eye of a subject comprising placingover the lens a contact lens comprising one or more zinc-containingcomponents, whereby the contact lens releases zinc ions onto the locusof the lens of the eye and/or adjacent tissues (such as muscles) in aneffective elastin-increasing amount. Such a lens, itself, also comprisesan aspect of this invention.

In other aspects, the invention comprises dermatologically and/orpharmaceutically acceptable compositions for the above methods andpurposes, containing, or consisting essentially of, an amount of one ormore zinc-containing components effective for the purpose in question,and also comprising one or more dermatologically and/or pharmaceuticallyacceptable carriers.

DETAILED DESCRIPTION OF THE INVENTION

As described briefly above, the invention comprises methods andcompositions for providing zinc, which may be in the form of zinc ions,to a selected site or portion of a subject's body in order to achieve afavorable result of the type described herein. A number of differentresults are contemplated herein, including increasing or decreasing fatcontent in skin or other tissues, increasing or decreasing epidermalthickness, and increasing elastin content. In general, as described inmore detail below, compositions containing one or more zinc-containingcomponents are applied, usually topically, to a site or location on thesubject's body that is deemed to be in need of one or more of the saidresults. By “in need” is meant both pharmaceutical or health-relatedneeds, for example, healing or restoring tissue, as well as cosmeticneeds, for example, altering or improving the appearance of tissue.

Compositions of the present invention are useful for regulating signs ofskin aging, particularly visible and/or tactile discontinuities in skintexture associated with aging. “Regulating the signs of skin aging”includes prophylactically regulating and/or therapeutically regulatingone or more of such signs (similarly, regulating a given sign of skinaging, e.g., lines, wrinkles or pores, includes prophylacticallyregulating and/or therapeutically regulating that sign). As used herein,prophylactically regulating such signs includes delaying, minimizingand/or preventing signs of skin aging. As used herein, therapeuticallyregulating such signs includes ameliorating, e.g., diminishing,minimizing and/or effacing signs of skin aging.

By “signs of skin aging” is meant outward visibly and tactilelyperceptible manifestations as well as other macro or micro effects dueto skin aging. These signs include the development of texturaldiscontinuities such as wrinkles, including both fine superficialwrinkles and coarse deep wrinkles, skin lines, crevices, bumps, largepores, scaliness, flakiness and/or other forms of skin unevenness orroughness, loss of skin elasticity, sagging (including puffiness in theeye area and jowls), loss of skin firmness, loss of skin tightness, lossof skin recoil from deformation, discoloration (including undereyecircles), blotching, sallowness, hyperpigmented skin regions such as agespots and freckles, keratoses, abnormal differentiation,hyperkeratinization, elastosis, collagen breakdown, and otherhistological changes in the stratum comeum, dermis, epidermis, the skinvascular system (e.g., telangiectasia or spider vessels), and underlyingtissues, especially those proximate to the skin.

Such signs may be induced or caused by intrinsic factors or by extrinsicfactors, e.g., chronological aging and/or environmental damage or otherpathological state. It should be noted, however, that in this embodimentthe present invention is not limited to regulation of theabove-mentioned signs of skin aging that arise due to mechanisms thatare associated with skin aging, but is intended to include regulation ofsaid signs irrespective of the mechanism of origin.

Compositions, devices and methods of this invention are useful fortherapeutically regulating visible and/or tactile discontinuities inmammalian skin texture, including texture discontinuities related toskin aging. This includes ameliorating, e.g., diminishing, minimizingand/or effacing visible and/or tactile discontinuities in the texture ofmammalian skin, to thereby provide improved skin appearance and/or feel,e.g., a smoother, more even appearance and/or feel. For example, thelength, depth, and/or other dimension of lines and/or wrinkles may bedecreased, the apparent diameter of pores may decrease, or the apparentheight of tissue immediately proximate to pore openings may decrease soas to approach that of the interadnexal skin.

The present invention is also useful for prophylactically regulatingvisible and/or tactile discontinuities in mammalian skin texture,including texture discontinuities associated with skin aging, that is,delaying, minimizing and/or preventing visible and/or tactilediscontinuities in the texture of skin, to thereby provide improved skinappearance and/or feel, e.g., a smoother, more even appearance and/orfeel.

Compositions of this invention contain one or more zinc-containingcomponents that are effective in carrying out the purposes. Theparticular zinc-containing component or components employed, and theconcentration in the compositions, can depend on the purpose for whichthe composition is to be applied.

In general, the zinc-containing components used in this inventioninclude ingredients such as compounds, complexes, chelates, etc. ofzinc. Among zinc compounds, particularly useful in the compositions andmethods of this invention are zinc salts, including acetate, ascorbate,aspartate, butyrate, caproate, caprylate, carbonate, chromate,citraconate, citramalate, citrate, EDTA, formate, fumarate, gallate,gluconate, halides, iodate, lactate, laurate, laureate, malate, maleate,malonate, metaphosphate, methanesulfonate, monophosphate, myristate,nitrate, octoate, oleate, orotate, orthophosphate, oxalate, oxides,palmitate, permanganate, phenolsulfonate, phosphate, picolinate,propionate, pyrophosphate, salicylate, selenate, stearate, succinate,sulfate, sulfonate, tannate, tartrate, tetrametaphosphate, titanate,transferrin, tripolyphosphate, undecylate, and valerate. Also, usable inthe invention are chelates of zinc and other types of zinc-containingchemical substances such as complexes, for instance complexes of zincwith amino acids such as methionine or nucleotide-based carriers and thelike.

The compositions of this invention are preferably in the form ofproducts to be applied to the skin, gums, eyes, or other tissues ofhumans or other mammals. They therefore contain a dermatologically orpharmaceutically acceptable carrier, vehicle or medium, i.e. a carrier,vehicle or medium that is compatible with the tissues to which they willbe applied. The term “dermatologically or pharmaceutically acceptable,”as used herein, means that the compositions or components thereof sodescribed are suitable for use in contact with these tissues or for usein patients in general without undue toxicity, incompatibility,instability, allergic response, and the like.

In addition, compositions of the invention may comprise any ingredientconventionally used in the fields under consideration, and particularlyin cosmetics and dermatology.

The term “effective amount” as used herein means an amount of a compoundor composition according to this invention that is sufficient tosignificantly induce a particular positive benefit, but that implicitlyis a safe amount, i.e. one that is low enough to avoid serious sideeffects. The positive benefit may be health-related, or it may be morecosmetic in nature, or it may be a combination of the two.

In terms of their physical form, compositions of this invention mayinclude solutions, emulsions (including microemulsions), suspensions,creams, lotions, gels, powders, or other typical solid or liquidcompositions used for treatment of skin, gums, eyes, etc. Suchcompositions will contain, in addition to the zinc salts of thisinvention, other ingredients typically used in such products, such asantimicrobials, moisturizers and hydration agents, penetration agents,preservatives, emulsifiers, natural or synthetic oils, solvents,surfactants, detergents, gelling agents, emollients, antioxidants,fragrances, fillers, thickeners, waxes, odor absorbers, dyestuffs,coloring agents, powders, viscosity-controlling agents and water, andoptionally including anesthetics, anti-itch actives, botanical extracts,conditioning agents, darkening or lightening agents, glitter,humectants, mica, minerals, polyphenols, silicones or derivativesthereof, sunblocks, vitamins, and phytomedicinals.

In addition to zinc-containing components, the compositions may containother active ingredients used in skin or dental care. For instance, theymay contain anti-acne actives; anti-wrinkle, anti-skin atrophy or skinrepair actives; skin barrier repair actives; non-steroidal cosmeticsoothing actives; artificial tanning agents and accelerators; sunscreenactives; sebum stimulators; sebum inhibitors; anti-oxidants; proteaseinhibitors; skin tightening agents; anti-itch ingredients; desquamatingenzyme enhancers; anti-glycation agents; and mixtures of such actives.

Compositions according to this invention may be in the form ofcontrolled-release or sustained-release compositions, wherein thezinc-containing component or components is encapsulated or otherwisecontained within a material such that it is released into thesurrounding environment (e.g. skin or other tissue) in a controlledmanner over time. The zinc-containing component or components may becontained within matrixes, liposomes, vesicles, microcapsules,microspheres and the like, or within a solid particulate material, allof which is selected and/or constructed to provide release of thezinc-containing component over time. The release-controlling materialmay be biodegradable or non-biodegradable, and may be applied per se orin the form of a composition such as an emulsion, suspension, cream,ointment, etc.

The compositions of the invention preferably are formulated so that theycontain an amount of one or more zinc-containing components that iseffective per se in obtaining the desired effect or result, as describedbelow, so that this desired effect can be obtained with a singleapplication, or with a relatively small number of repeated applications.In general, the compositions of this invention typically contain a totalconcentration of the zinc-containing component or components that isfrom about 1.0 picomolar (pM) to about 100 millimolar (mM). Thecompositions are applied over a period of time, and in a number ofapplications, so as to achieve the particular objective desired. In somecases the ranges over which particular effects occur are different whilein others (for instance in the case of fat alteration and elastindeposition) an overlap exists. The examples herein contain guidance ofthe effects obtained by using different concentrations of zinc compoundsor complexes and varying frequencies and lengths of application of thecompositions. If one effect is desired, a concentration at which thateffect predominates would be selected. An interesting range for topicalapplication is one that accomplishes epidermal thickening and increaseselastin content together with either an increase in fat content or nodecrease in fat content. To achieve this goal, the ranges and durationsof topical applications in the examples may be used. In some cases theapplication of such a composition will be carried out by or under thesupervision of a dispensing physician, chiropractor or other health careprofessional, who will be responsible for ensuring that the properamount of the composition is used to achieve the desired effect.

Elasticity and Elastin.

In one aspect, the invention provides methods, compositions and devicesfor improving tissue elasticity or increasing elastin levels. In oneembodiment the invention comprises a method for increasing elastincontent in a tissue of a subject (including but not limited to theskin), which comprises topically applying to said tissue adermatologically or pharmaceutically acceptable composition consistingessentially of one or more zinc-containing components in an effectiveelastin-increasing amount in admixture with at least onedermatologically or pharmaceutically acceptable carrier. Thisapplication is particularly relevant for increasing skin elasticity orelastin levels either to reverse aging-related or other undesirablechanges or to provide a cosmetic improvement.

Areas of the skin to which the compositions may be applied include theface (including cheeks, peri-eye, circum-oral, forehead, lips andperi-nose), breasts, buttocks, neck, arms, legs, torso, or furrows orwrinkles in the face, hands and neck.

For improving elastin or elasticity in the skin, a composition accordingto this invention contains one or more zinc-containing components in atotal concentration of from about 1.0 picomolar (pM) to about 900 μM,preferably from about 100 to about 500 pM. The composition may beapplied topically so as to provide an effective amount of zinc to thearea where the effect is desired, and may be applied at varyingintervals and over varying durations to achieve the desired degree ofincrease in elastin content.

In another embodiment of the invention, a contact lens, preferably ahard lens, coated with one or more zinc-containing components isemployed for the purpose of altering the elasticity of the lens of theeye so as to change its shape and/or render it more elastic. Suchapplication may be carried out alone or in conjunction with othertreatments that are aimed at improving vision, for example treatments todiminish presbyopia. Coating the lens may be done, for instance, usingtechniques mentioned in U.S. Pat. Nos. 6,113,636, 6,190,407, 6,267,782and 6,322,588. The contact lens could be treated with a plasma dischargeso that the outer margins of its inner surface would be derivatized (forexample with vaporized allylamine). This reactive surface, which wouldcontact the outer margins of the lens when properly used, would then belinked to a chelator of variable affinity for zinc ions, preferably onehaving 6 or more carbon atoms for controlled release of ionic zinc (forexample, undecylenate). This linkage either would be carried out in thepresence of zinc ions or would be followed by loading of the contactwith ionic zinc prior to administration. Alternatively, thezinc-containing coating may first be applied to the material from whichthe lens will be made, as discussed generally in these patents.

In this embodiment, the contact lens would produce transient reshapingof the lens of the eye, and the ionic zinc would induce elastinproduction in that lens to confer elasticity for the corrected shape.The lens of the eye would tend to retain the corrected shape even afterremoval of the contact lens. Treated contact lenses for this embodimentof the invention preferably produce local ionic zinc concentrations from0.1 to 100 micromolar in the vicinity of the lens. Within the effectiverange, lower concentrations of zinc may be used over longer periods oftime to achieve the same result, or higher concentrations may be usedfor shorter periods. Generally, the zinc concentration may range fromabout 1.0 pM to about 500 mM of the zinc-containing component orcomponents, preferably from about 100 pM to about 50 mM, at thetissue-contact lens interface.

Alteration of Fat Levels.

In another embodiment of the invention, compositions of the inventionare applied topically to alter local fat levels either by increasing thefat level or by decreasing it. This alteration of fat level is believedto be due to an alteration of local zinc levels. The fat level affectedmay be that of the skin itself (“superficial fat”) or may be of tissuelying below the skin (“deep fat”). Such application can be made alone orin combination with other factors to either increase or decrease localfat levels. Methods according to the invention for increasing fatprovide benefits such as cosmetic benefits in reducing appearance offurrows or deep wrinkles, breast augmentation (contour or size) throughtopical application, lip augmentation for cosmetic benefit, soft tissuereconstruction or remodeling for cosmetic benefit, or tissuereconstruction of a contour defect to reduce the appearance of thedefect, or to provide desired cosmetic or functional effects. Methodsfor decreasing fat provide benefits such as reduced trunk, leg or otherfat stores to decrease apparent weight or improve gross aestheticcontour (e.g., topical pseudo-liposuction), decreased volume ofperi-orbital fat (e.g., topical pseudo-blepharoplasty), or lipreduction.

For increasing fat the compositions may be applied to areas of the bodysuch as the lips, soft tissue, furrows or wrinkles in the face, breasts,stretch marks, buttocks, cheeks, arms, and/or legs. For decreasing fatthe compositions may be applied to the torso (including the anteriorabdomen or belly), isolateral abdomen (commonly known as “lovehandles”), legs, face, neck, buttocks, arms, legs and eyelids or theperi-eye region.

Compositions for use in this embodiment of the invention for increasingfat content contain one or more zinc-containing components, in a totalconcentration of from about 1.0 pM to about 900 μM, preferably fromabout 100 pM to about 500 μM. Compositions for decreasing fat contentcontain the zinc-containing component or components in a higher totalconcentration, one of from about 10 μM to about 100 mM, preferably fromabout 100 μM to about 10 mM.

Changing Epidermal Thickness.

Topical application of a composition according to this invention can becarried out for the purpose of increasing epidermal thickness, forinstance, to decrease appearance of fine lines or wrinkles, or toreverse epidermal atrophy from age or various pathologic states. Suchapplications can be made to appropriate parts of the body, particularlythe face (areas adjacent to the eyes, lips or forehead, for example).Conversely, compositions of this invention may be applied to decreaseepidermal thickness, for instance, to decrease or eliminate scars andstretch marks. Again, these effects are believed to occur as a result ofan alteration of zinc levels in the relevant tissues.

Compositions for increasing epidermal thickness will contain one or morezinc-containing components in a total concentration of from about 1.0 pMto about 900 μM, preferably from about 100 pM to about 500 Compositionsfor decreasing epidermal thickness will contain a total concentration offrom about 10 μM to about 100 mM zinc-containing components, preferablyfrom about 100 μM to about 10 mM.

Gum Treatments.

Compositions of this invention may also be applied topically to thegums, to increase tissue zinc levels in order to prevent or treat gumregression or atrophy. This is believed to result from the effect of thezinc in promoting elastin increase and/or the increase of epidermaltissue, a possible combination of two effects, to better anchor theteeth in place. Compositions for this purpose will contain a totalconcentration of from about 1.0 pM to about 100 mM, preferably fromabout 100 pM to about 900 μM, of one or more zinc-containing components.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters that could be changed or modified to yieldessentially similar results.

Example 1 Topical Application of a Zinc Formulation to Increase SkinElasticity

To achieve a therapeutic benefit for skin elasticity, initial stocksolutions were prepared as follows:

Sample Stock Solutions

-   -   a) Base only (“blank”)    -   b) 10 μM Zn⁺⁺ (“Z low”)    -   c) 1.0 mM Zn⁺⁺ (“Z med”)    -   d) 100 mM Zn⁺⁺ (“Z high”)

To prepare these solutions, a 1.0 M stock of zinc acetate [ZnAc*(H2O)₂](Mallinkrodt Lot 8740 KPRK) was prepared in sterile phosphate bufferedsaline (PBS, pH 7.2, Gibco BRL Lot #1111327) by dissolving 109.75 g in500 mL. To 0.2 ml of Cetaphil® moisturizer base was added 20 μl ofsolution a, b, c, or d depending upon group (corresponding to Groups“A,” “B,” “C,” or “D”). For group A, added 20 μl PBS alone to 180 μlCetaphil×84 tubes. For group B, added 20 μl 1.0M Zn to 180 μlCetaphil×84 tubes. For group C, added 200 μl of 1.0M Zn to 19.8 ml PBS,vortexed, labeled as 0.01M Zn, and added 20 μl of 1.0M Zn to 180 μlCetaphil×84 tubes. For group D, added 200 μl 0.01 M Zn to 19.8 ml PBS,vortexed, labeled as 0.1 mM Zn, and added 20 μl 1.0M Zn to 180 μlCetaphil×84 tubes. After addition of samples to moisturizer base, thesamples were mixed to homogeneity and stored at 4° C. overnight. At N=4per treatment group, C57 Black Six mice at 8 weeks of age wereanesthetized with 3% isoflurane by inhalation, and were shaved atmid-scapular dorsal region. Moisturizer was applied daily at 0.2 cc perday for each group for a 21 day period. After 21 days application, thetreated skin segment was harvested en bloc and subdivided into threeequal portions: a cranial portion, a left lateral portion, and a rightlateral portion. The cranial portion and the left lateral portion werefixed in 10% neutral buffered formalin for 12-16 hours, then rinsed in70% ethanol and embedded in paraffin. The right lateral portion was snapfrozen in OCT medium at the time of harvest and stored at −35° C. untiluse.

Paraffin-embedded specimens were sectioned at 4-6 microns,deparaffinized, and stained with Verhoeff elastica stain formorphological assessment of elastin content. All procedures and analyseswere performed by blinded observers. High resolution digital micrographsof each preparation were obtained using a Diagnostic Instruments SPOTcamera (Diagnostic Instruments, Sterling Heights, Mich.) as displayed ona Nikon E600 epifluorescence microscope with plan-apochromat lenses.Representative photomicrographs depicting elastin content (black fibers)are depicted as FIGS. 1( a)-1(d).

FIG. 1 depicts representative photomicrographs of murine skin sectionedand stained with Verhoeff Elastica stain after 21 days topical treatmentwith a) Base only (“blank”), b) 10 μM Zn++ (“Z low”), c) 1.0 mM Zn++ (“Zmed”), or d) 100 mM Zn++ (“Z high”) depicting increasing elastin levels.As the dose of Zn increases from zero (a) to low (b) to medium (c) tohigh (d) the length, density and thickness of the black elastic fibersincreases significantly. At high dose, epidermal sloughing andirritation occurs, however. Lower doses afford the benefits withoutlocal signs of irritation. Overall, ionic zinc affords dose-dependantincreases in the elastin content of skin after topical administration.

Example 2 Topical Application of a Zinc Formulation to Alter EpidermalThickness

Paraffin-embedded specimens from animals treated in example 1 above weresectioned at 4-6 microns, deparaffinized, and stained with a combinationof Verhoeff elastica stain and the Masson trichrome stain formorphological assessment of epidermal thickness. All procedures andanalyses were performed by blinded observers. High resolution digitalmicrographs of each preparation were obtained using a DiagnosticInstruments SPOT camera (Diagnostic Instruments, Sterling Heights,Mich.) as displayed on a Nikon E600 epifluorescence microscope withplan-apochromat lenses. Images were analyzed using Image Pro Plussoftware (Media Cybernetics, Silver Spring, Md.) to determine totalcross-sectional area of epidermis over standardized lengths. Mean andstandard error were assessed using Statview (Abacus Concepts, Berkeley,Calif.), with comparisons made using ANOVA repeated measures andsignificance determined at 95% with post-hoc testing using Fisher PLSDor Scheffeiii F-test. Results are presented as Table 1 below.

TABLE 1 Epidermal area (pixels) across standardized epidermal lengths 21days after treatment with a) Base only (“blank”), b) 10 mM Zn++ (“Zlow”), c) 1.0 mM Zn++ (“Z med”), or d) 100 mM Zn++ (“Z high”). GroupMean Std. Error Base only (“blank”) 18221.646 600.673 10.0 μM Zn⁺⁺ (“Zlow”) 16786.125 676.14 1.0 mM Zn⁺⁺(“Z med”) 20799.188 653.073 100 mMZn⁺⁺(“Z high”) 27365.292 890.926

Paraffin-embedded specimens from animals were treated in Example 1except that the doses of 10.0 μM Zn⁺⁺, 1.0 μM Zn⁺⁺, 0.1 μM Zn⁺⁺, and thecontrol were applied for 42 days. Specimens were fixed and embedded asabove, sectioned at 4-6 microns, deparaffinized, and stained with acombination of Verhoeff elastica stain and the Masson trichrome stainfor morphological assessment of epidermal thickness. All procedures andanalyses were performed by blinded observers. High resolution digitalmicrographs of each preparation were obtained using a DiagnosticInstruments SPOT camera (Diagnostic Instruments, Sterling Heights,Mich.) as displayed on a Nikon E600 epifluorescence microscope withplan-apochromat lenses. Images were analyzed using Image Pro Plussoftware (Media Cybernetics, Silver Spring, Md.) to determine totalcross-sectional area of epidermis over standardized lengths. Mean andstandard error were assessed using Statview (Abacus Concepts, Berkeley,Calif.), with comparisons made using ANOVA repeated measures andsignificance determined at 95% with post-hoc testing using Fisher PLSDor Scheffe F-test. Results are presented as Table 2 below.

TABLE 2 Epidermal area (pixels) across standardized epidermal lengths 42days after treatment with a) Base only (“blank”), b) 10 μM Zn++ (“Zlow”), c) 1.0 μM Zn++ (“Z med”), or d) 0.1 μM Zn++ (“Z high”). P =0.0014 by ANOVA repeated measures. Group Mean Std. Error Base only(“blank”) 6728.286 143.808 10 μM Zn⁺⁺ (“Z high”) 7203.875 230.472 1.0 μMZn⁺⁺(“Z med“) 7724.190 240.195 0.1 μM Zn⁺⁺(“Z low”) 6710.000 173.975

Example 3 Topical Application of a Zinc Formulation to Decrease Fat(Hypodermal Brown Fat)

Paraffin-embedded specimens from animals treated in Example 1 above weresectioned at 4-6 microns, deparaffinized, and stained with a combinationof Verhoeff elastica stain and the Masson trichrome stain formorphological assessment of brown adipose tissue area. All proceduresand analyses were performed by blinded observers. High resolutiondigital micrographs of each preparation were obtained using a DiagnosticInstruments SPOT camera (Diagnostic Instruments, Sterling Heights,Mich.) as displayed on a Nikon E600 epifluorescence microscope withplan-apochromat lenses. Images were analyzed using Image Pro Plussoftware (Media Cybernetics, Silver Spring, Md.) to determine totalcross-sectional area of epidermis over standardized lengths. Mean andstandard error were assessed using Statview (Abacus Concepts, Berkeley,Calif.), with comparisons made using ANOVA repeated measures andsignificance determined at 95% with post-hoc testing using Fisher PLSDor Scheffe F-test. Results are presented as Table 3 below (P=0.0001).

TABLE 3 Cross-sectional hypodermal adipose area (pixels) acrossstandardized epidermal lengths 21 days after treatment with a) Base only(“blank”), b) 10 mM Zn++ (“Z low”), c) 1.0 mM Zn++ (“Z med”), or d) 100mM Zn++ (“Z high”). Mean Standard Error a) Base only (“blank”)456566.375 19714.059 b) 10 μM Z⁺⁺ (Z low) 436809.417 20948.052 c) 1.0 mMZn⁺⁺ (Z med) 377185.833 33645.848 d) 100 mM Zn⁺⁺ (“Z high”) 132251.3335078.162

Since brown adipose tissue and white adipose tissue respond differentlyand since different combinations of growth factors alter responses foreach, ionic zinc can be applied to either significantly increase ordecrease fat area, as illustrated in this and the following example.

Example 4 Topical Application of a Zinc Formulation to Decrease Fat(Deep White Fat)

Stock zinc solutions were prepared as above and diluted in Cetaphil asabove to achieve a final concentration of 10 μM Zn⁺⁺ in moisturizerbase. After addition to moisturizer base, the samples were mixed tohomogeneity and stored at 4° C. overnight. At N=4 per treatment group,C57 Black Six mice at 8 weeks of age were anesthetized with 3%isoflurane by inhalation. Moisturizer was applied daily to the lateralcurvature of the abdomen from the ribs to the pelvis centered at themid-axillary line (“love handles”). Moisturizer was applied at 0.2 ccper day for each group for a 21 day period. After 21 days applicationthe treated skin segment was harvested en bloc full depth and fixed in10% neutral buffered formalin for 12-16 hours, then rinsed in 70%ethanol and embedded in paraffin.

Paraffin-embedded specimens were sectioned at 4-6 microns,deparaffinized, and stained with a combination of Verhoeff elasticastain and the Masson trichrome stain for morphological assessment ofbrown adipose tissue area. All procedures and analyses were performed byblinded observers. High resolution digital micrographs of eachpreparation were obtained using a Diagnostic Instruments SPOT camera(Diagnostic Instruments, Sterling Heights, Mich.) as displayed on aNikon E600 epifluorescence microscope with plan-apochromat lenses.Images were analyzed using Image Pro Plus software (Media Cybernetics,Silver Spring, Md.) to determine total cross-sectional area of epidermisover standardized lengths. Mean and standard error were assessed usingStatview (Abacus Concepts, Berkeley, Calif.), with comparisons madeusing ANOVA repeated measures and significance determined at 95% withpost-hoc testing using Fisher PLSD or Scheffe F-test. Results arepresented as Table 4 below (P=0.0001).

TABLE 4 Cross-sectional deep adipose area (pixels) across standardizedepidermal lengths 21 days after treatment with a) Base only (“blank”),b) 10 μM Zn++ as described. P = 0.0001 by ANOVA repeated measures(Staview SE, Abacus Concepts, Berkeley, CA) Mean Standard Error a) Baseonly (“blank”) 29375.425 937.893 b) 10 μM Zn⁺⁺ 73011.321 5060.653

Example 5 Local Application of a Zinc Formulation to Treat or PreventGum Regression

A gel or paste solution is used to release ionic Zn to the gum area. Forinstance, zinc acetate can be added to an ointment formulation. Anexample of this mixture would consist of Tween 80, glycerol starch,sterile deionized water and triethanolamine. The most preferredformulation would attain local ionic zinc concentrations of 1 to 100micromolars. This zinc oral composition would be used to treat gumregression and aid in tooth loss caused by gum disease and age relateddegeneration of gums. Increased elastin and epidermal tissue would helpto anchor the tooth in place and provide support.

Examples 6-8

Examples 6-8 show the effect of the application of a compositionaccording to the invention in (a) increasing tropoelastin content, (b)altering epidermal thickness, elastin content and hypodermal fat, and(c) increasing skin elasticity. Among others, it was found that ingeneral the effects of increasing epidermal thickness, increasingepidermal fat and inducing new elastin production are dose-dependent andthat the effective ranges of active ingredients for these three effectsoverlap.

Example 6 Topical Application of a Zinc Formulation to IncreaseTropoelastin Content

A stock zinc solution was prepared as above to achieve a finalconcentration of 100 micromolar Zn++ in commercial moisturizer bases.After addition to moisturizer bases, the samples were mixed homogeneityand stored at room temperature. At N=3 per group, C57 Black Six mice at8 weeks of age were anesthesized with 3% isoflurance by inhalation.Moisturizer was applied daily to the dorsal skin of the mouse.Moisturizer was applied at 0.2 cc per day for each group for a 7-dayperiod. After 7-day application, the treated dorsal skin was harvestedand divided into three segments. One segment fixed in 10% neutralbuffered formalin for 12-16 hours, then rinsed in 70% ethanol andembedded in paraffin. Two segments were frozen and stored at -73?C.

Paraffin-embedded specimens were sectioned at 4-6 microns,deparaffinized, and probed with fluorescein labeled DNA primers totropoelastin (5′FGCCTGGTGCCCACT and 5′FCTCCACCAAGGCCATA) and stainedwith Fast Red stain. Paraffin-embedded specimens were sectioned at 4-6microns, deparaffinized, and stained with antibody conjugated toalkaline phosphate and counterstained with Eosin stain. All proceduresand analyses were done by blinded observers. High resolution digitalmicrographs of each preparation we obtained using a DiagnosticInstruments SPOT camera (Diagnostic Instruments, Sterling Heights,Mich.) as displayed on a Nikon E600 epifluoresence microscope with aplan-apochromat lenses. Images were analyzed using Image Pro Discoverysoftware (Media Cybernetics, Silver Spring, Md.) to determine totalcross-sectional tropoelastin content. Mean and standard error wereassessed using Statview (Abacus Concepts, Berkeley, Calif.), withcomparison made using ANOVA repeated measure and significance determinedat 95% with post-hoc testing using Fischer PLSD or Scheffe F-test.

FIG. 2 shows specimens of the above, for comparison, with (A) being aspecimen of the control (no additional zinc compound) and (B) of theinvention. FIG. 3 depicts the increase in tropoelastin mRNA levels withtreatment by the zinc-containing composition (black bar). This increaseis paralleled by an increase in newly translated (hence soluble)tropoelastin by immunohistochemistry.

Example 7 Topical Application of a Zinc Formulation to Alter EpidermalThickness, Elastin Content, and Hypodermal Fat

A stock zinc solution was prepared and applied as above in example 6.

Paraffin-embedded specimens were sectioned at 4-6 microns,deparaffinized, and stained with Hematoxylin and Eosin stain. Allprocedures and analyses were done by blinded observers.Semi-quantitative scoring was done to measure cross-sectional epidermalthickness, elastin content, and hypodermal fat.

FIGS. 4, 5 and 6 show comparative scores for elastin content, epidermalthickness, and hypodermal fat, using semi-quantitative scales.

Example 8 Topical Application of a Zinc Formulation to Increase SkinElasticity

Stock zinc solution was prepared as above to achieve a finalconcentration of 100 micromolar Zn++ in commercial moisturizer base.After addition to moisturizer bases, the samples were mixed homogeneityand stored at room temperature. Moisturizer was applied twice daily tothe peri- and infra-orbital eye region. At baseline and each 7 dayperiod later, video images were captured using Canon ZR60 (Canon,Jamesburg, N.J.). Snap time was determined as a measure of compositeskin elasticity using Abode AfterEffects software.

FIGS. 7-11 show the results of tests on five human subjects treated forup to six weeks, as indicated.

Example 9 Effective Zinc Concentrations for Induction of ElastinProduction

A stock zinc solution was prepared as above to achieve a finalconcentration of 100 micromolar Zn++ in commercial moisturizer bases.After addition to moisturizer bases, the samples were mixed homogeneityand stored at room temperature. At N=4 per group, C57 Black Six mice at8 weeks of age were anesthesized with 3% isoflurance by inhalation.Moisturizer was applied daily to the dorsal skin of the mouse.Moisturizer was applied at 0.2 cc per day for each group for a 21-dayperiod. After 21-day application, the treated dorsal skin was harvestedand divided into three segments. One segment fixed in 10% neutralbuffered formalin for 12-16 hours, then rinsed in 70% ethanol andembedded in paraffin. Two segments were frozen and stored.

Paraffin-embedded specimens were sectioned at 4-6 microns,deparaffinized, and stained with verhoeff-van giesson elastica stain.All procedures and analyses were done by blinded observers. Highresolution digital micrographs of each preparation we obtained using aDiagnostic Instruments SPOT camera (Diagnostic Instruments, SterlingHeights, Mich.) as displayed on a Nikon E600 epifluoresence microscopewith a plan-apochromat lenses. Images were analyzed using Image ProDiscovery software (Media Cybernetics, Silver Spring, Md.) to determinetotal cross-sectional elastin content as % of total. Mean and standarderror were assessed using Statview (Abacus Concepts, Berkeley, Calif.),with comparison made using ANOVA repeated measure and significancedetermined at 95% with post-hoc testing using Fischer PLSD or ScheffeF-test. Test results are shown in FIG. 12 (G—control; H—zinc-containingcomposition), which depicts cross-sectional elastin content after a21-day topical application of zinc or control.

It also has been found that zinc, for example, zinc acetate, acts bystabilizing, then destabilizing, then restabilizing elastase function asthe concentration of zinc-containing components increases. This patternof activity likely contributes to floor and ceiling concentrationsobserved for induction of elastin production.

An assay was run that involved varying concentrations of each divalentcation, ranging from concentrations of 0.005M to 1.0M, in phosphatebuffered saline (PBS). The positive control contained no cations, whilethe negative control contained no elastase. Solutions were read at 405nm every minute for 15 minutes under controlled conditions. The opticaldensity value of the negative control, which represented backgroundinterference, was subtracted from each result. These corrected opticaldensity values from the two trials were averaged.

Elastase activity increased for all the ions Mg²⁺, Ca²⁺, and Zn²⁺between the interval of 0.005M to 0.01M cation concentration. At allcation concentrations greater than 0.01M, elastase activity sharplydecreased, and higher concentrations continued to result in decreasingelastase activity toward a minimum value. Divalent cations were found toinduce elastase activity at very low concentrations (<0.01M), followedby a range of concentrations where the presence of divalent cationsinhibited elastase (0.01M˜0.75M) to a minimum value greater than zero.At very high concentrations, divalent cations were found to once againinduce elastase activity (>0.75M).

Elastase was found to be most sensitive to Ca²⁺ ions (the most potentimmediate inhibitor) while most constantly inhibited by Zn²⁺ as timeprogressed. Elastase was least sensitive to Mg²⁺. The results are shownin FIG. 13.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1-35. (canceled)
 36. A method for increasing epidermal thickness in asubject comprising topically applying a composition consistingessentially of one or more zinc-containing components in admixture witha dermatologically or pharmaceutically acceptable carrier, in aneffective epidermal-thickness-increasing amount, to a site of the skinof the subject in need of increased epidermal thickness.
 37. A methodaccording to claim 36 wherein the composition is topically applied toone or more areas in the face that have fine lines or wrinkles, and/orto other areas of the skin exhibiting epidermal atrophy due to age ordue to an underlying pathological state.
 38. A method according to claim36 wherein the composition comprises one or more zinc compounds.
 39. Amethod according to claim 36 wherein the composition comprises one ormore zinc salts.
 40. A method according to claim 39 wherein the one ormore zinc salts is selected from the group consisting of zinc acetate,ascorbate, aspartate, butyrate, caproate, caprylate, carbonate,chromate, citraconate, citramalate, citrate, EDTA, formate, fumarate,gallate, gluconate, halides, iodate, lactate, laurate, laureate, malate,maleate, malonate, metaphosphate, methanesulfonate, monophosphate,myristate, nitrate, octoate, oleate, orotate, orthophosphate, oxalate,oxides, palmitate, permanganate, phenolsulfonate, phosphate, picolinate,propionate, pyrophosphate, salicylate, selenite, stearate, succinate,sulfate, sulfonate, tannate, tartrate, tetrametaphosphate, titanate,transferrin, tripolyphosphate, undecylate, and valerate, and mixturesthereof.
 41. A method according to claim 40 wherein the compositioncomprises zinc acetate.
 42. A method according to claim 36 wherein thecomposition comprises one or more zinc chelates.
 43. A method accordingto claim 36 wherein the composition comprises one or more zinccomplexes.
 44. A method according to claim 36 wherein the carrier is adermatologically acceptable carrier and further comprises a moisturizer.45. A method according to claim 36 wherein the one or morezinc-containing component is present in the composition in aconcentration of from about 1.0 pM to about 900 μM.
 46. A methodaccording to claim 36 wherein the one or more zinc-containing componentis present in the composition in a concentration of from about 100 pM toabout 500 μM. 47-98. (canceled)
 99. A method for increasing epidermalthickness a region of skin of a subject that is in need of increasedepidermal thickness, the method consisting essentially of applyingtopically a composition consisting essentially of one or morezinc-containing components in admixture with a dermatologicallyacceptable carrier to the region of skin of the subject, wherein the oneor more zinc-containing components is selected from the group consistingof zinc acetate, zinc ascorbate, zinc aspartate, zinc butyrate, zinccaproate, zinc caprylate, zinc carbonate, zinc citraconate, zinccitramalate, zinc citrate, zinc EDTA, zinc formate, zinc fumarate, zincgallate, zinc gluconate, zinc halides, zinc lactate, zinc malate, zincmaleate, zinc malonate, zinc metaphosphate, zinc monophosphate, zincnitrate, zinc octoate, zinc orotate, zinc orthophosphate, zinc oxalate,zinc phosphate, zinc picolinate, zinc propionate, zinc pyrophosphate,zinc salicylate, zinc selenate, zinc succinate, zinc sulfate, zincsulfonate, zinc tartrate, zinc tetrametaphosphate, zinctripolyphosphate, zinc valerate, zinc amino acid complexes, zincnucleotide complexes, and mixtures thereof, wherein zinc of the said oneor more zinc-containing components is present in the composition at aconcentration that increases epidermal thickness without causingirritation due to zinc, wherein said concentration is in the range of1.0 pM to 900 μM, and wherein the epidermal thickness in the region ofskin is increased.